Transcript Microbiology 155

Microbiology 155
Lecture 2- Microscopy
Microscopy
Properties of light
• Wavelengths of light= colors. The
visible spectrum
•Ranges from 420-680 nm
Resolution of light
The resolution which is the ability to
see items as separate and distinct
entities is determined by the
wavelength of light used in the
microscope
Resolution and Images
•For two points to be seen as separate
the light has to be able to pass between
them
•If the points are very close and the
light is not able to pass between, the
image will appear as fuzzy.
•The light microscope used in class is
unable to resolve two points that are
closer together than 220 nm
• The average wavelength of light is 550
nm
The Resolving Power of a
Lens
•The resolving power of a lens is a
numerical measure of the resolution
that can be attained with that lens.
• The smaller the distance between two
points that can be resolved the stronger
the power of the lens
•Ultra violet light has a wavelength of
100- 400 nm, As a result It can resolve
points that are 110 nm apart.
The Numerical Aperture ( NA)
•NA= the numerical apertures . The numerical
aperture is a measure of the light that is
collected and directed through the microscope
oculars. The part of the microscope that
collects the light is referred to as the
condenser.
•RP= Lambda/2NA
Reflection
• If light strikes an object and bounces
back- reflection has occurred
Refraction
• If light bends as it passes from one
medium to another - through different
densities- it is bent or refracted
•The index of refraction for a material is
the measure of the speed at which the light
passes through the material.
Bright Field Microscopy
( used to visualize stained
slides of bacteria
Dark Field
A microscope adapted for
dark field has a condenser
that prevents light from
being transmitted through
the specimen on the slide.
The background appears
dark and the bacterium or
organism appears to glow
Used to show contrast such as with
spirochetes.
Phase Contrast Microscopy
This microscopic technique is used
to view live cells. It amplifies
differences in the cellular structure
and contents. These are called
refractive differences .
Phase Contrast Microscopy- Images
NOMARSKI DIFFERENTIAL INTERFERENCE
• Operate like phase contrast microscopes but with
much greater resolution
• This produces almost a three dimensional image
Fluorescence Microscopy
Fluorescent stains use colored
molecules that become
excited when short wave
lengths of light shine on them
These colored molecules
are called fluorochromes
Fluorescence- Antibody Staining
•Antibodies are molecules produced by the
immune system in response to invaders such
as bacteria, protozoans, and viruses.
• Antigens are foreign substances that are
present
•An antigen- antibody reaction is a specific
reaction
• This can be linked to the fluorescent
staining
Electron Microscopy
• Use electron beam as light source
• Electrons have shorter wavelength than
visible light
• Electromagnetic lenses help to magnify and
focus
• Resolution improves to .2um
• Photographs can be taken although objects
can not be directly visualized these are known
as
• Electron micrographs
Transmission Electron
Microscopy
TEM
• The electron beam is concentrated and passed
through an object
• The object is stained with heavy metal atoms
• The interaction of the electrons with the object
produces an image
• It is a flat image
Transmission Electron Microscopy showing the
internal structure. These cells are dividing by
the process of fission
Scanning Electron Microscopy- SEM
•In scanning electron microscopy the cells
are stained on the outside. The electron
beams are shot at the cells and bounce
back.
•They are caught by a recording device that
transmits the electrons to a TV screen
•The image can be visualized on the screen
Electron Micrograph of Blood Cells
These cells are reacting to the presence of
bacteria in the blood stream.
Freeze Etching and Freeze
Fracture
Cells are frozen in liquid nitrogen
The cells are broken open or fractured by
touching with a knife
The inner membranes and structures of
the cell are revealed “ moonscape”
In freezing etching - the surface of the
“moonscape” is layered with a heavy metalThe surface is then viewed with an
electron microscope
Freeze Etching of Yeast
Stains
• Stains have special characteristics
that allow them to bind to the cell wall
of bacteria
• Bacterial cell walls are negative.
• Bacterial stains are positively
charged
• Stains that are positively charged
are cationic
Stain terminology
• Simple stain- contains one dye molecule
• Differential stains are composed of two or
more dyes
• Negative stains color the background
• Flagellar stains add layers of dye or metal
to the surface of flagella
Visualization of Bacterial Cells
Staining Protocols
Stain components of cell walls
Gram - These bacteria stain pink with the
gram stain
Gram + These bacteria stain purple with
the gram stain
Gram Stain Protocol
• Smear slide with cells from culture of
bacteria
• Heat fix. Move slide through the flame
• Cover smear with Crystal Violet( Purple
Stain)- 1 min
• Rinse with water. Allow water to rinse
purple stain off the slide
• Cover the slide with Iodine. Iodine is a
mordant. It helps cells sticks to slide
Gram Stain ( continued)
• Place a few drops of alcohol on the slide
• This will remove any stain that is not
permanently attached to the cell walls
• Place drops of safranin over smear
• Rinse and air dry
Gram Staining
Acid Fast Staining- specialized technique
Acid fast bacteria retain carbol fuschin
and appear red
Non- acid fast accept the methylene
blue counterstain and appear blue
Mycobacterium is identified with this
staining procedure
Leprosy and tuberculosis
Acid fast staining- arrows pointing to
acid fast bacteria( reddish)
Spore staining- important to bacteria
called spore formers
clostridium,botulism, tetanus, and anthrax
Endospores retain malachite green stain
Vegetative cells accept safranin and
appear red
Schaeffer-Fulton spore stain- makes
spores easier to visualize